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Identification of SUMO-modified proteins by affinity purification and tandem mass spectrometry in Arabidopsis thaliana

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Abstract

Reversible conjugation of the small ubiquitin modifier (SUMO) peptide to proteins (SUMOylation) plays important roles in cellular processes in eukaryotes. Although more than 200 SUMO target proteins have been characterized in yeasts and animals, only very few SUMO targets are known in plants. Here, to identify putative SUM1 associating proteins in Arabidopsis we developed a proteomics-type protocol employing a His-GST-AtSUM1 column conjugated to Affi-gel beads with total protein extracts from 3–4 week old seedlings. AtSUM1 binding proteins and/or sumoylated targets were obtained by a change in pH of the eluant and identified by mass spectrometry. Fifteen candidate SUMO-modified proteins have so far been identified. Candidate proteins were analyzed by a split ubiquitination assay in yeast and additionally by split-luciferase complement assays in planta to confirm binding specificity and activity to AtSUM1, and productive sumoylation through in vivo assays in E. coli. Among proteins found reliably sumoylated are AtMYB31 (transcription factor), ACS7 (1-amino-cyclopropane-1-carboxylate synthase 7), and Rho GDI1 (Rho GDPdissociation inhibitor family protein). These proteins will provide a basis for studying functions of SUMOylation in the regulation of diverse processes in Arabidopsis.

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Authors and Affiliations

  1. Division of Applied Life Science (BK21 program), and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 660-701, Korea

    Hee Jin Park, Hyeong Cheol Park, Wonkyun Choi, Woo Sik Chung & Dae-Jin Yun

  2. Laboratory of Cytokine Immunology, Department of Biomedical Science and Technology, Konkuk University, Seoul, 143-701, Korea

    Jida Choi & Soohyun Kim

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  1. Hee Jin Park
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  2. Hyeong Cheol Park
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  3. Jida Choi
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  4. Wonkyun Choi
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  5. Woo Sik Chung
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  6. Soohyun Kim
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  7. Dae-Jin Yun
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Correspondence to Dae-Jin Yun.

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These authors contributed equally to this work.

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Park, H.J., Park, H.C., Choi, J. et al. Identification of SUMO-modified proteins by affinity purification and tandem mass spectrometry in Arabidopsis thaliana . J. Plant Biol. 56, 176–185 (2013). https://doi.org/10.1007/s12374-013-0127-1

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  • DOI: https://doi.org/10.1007/s12374-013-0127-1

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